Antimicrobial properties of promising Zn–Fe based layered double hydroxides for the disinfection of real dairy wastewater effluents

Bacterial resistance to conventional antibiotics is a serious challenge that requires novel antibacterial agents. Moreover, wastewater from dairy farms might contain countless number of pathogens, organic contaminants and heavy metals that consider a threat to the terrestrial and aquatic environment. Therefore, the development of cost-effective, highly operation-convenient, recyclable multifunctional antimicrobial agents became an urgent necessity. Layered double hydroxides (LDH) have shown promising results as antibacterial agents. However, more work is required to further investigate and improve the antimicrobial performance of LDH structures against pathogens. In this study three Zn–Fe based LDH were investigated for real dairy wastewater disinfection. The three LDH samples were cobalt substituted Zn–Fe LDH (CoZnFe), magnesium substituted Zn–Fe LDH (MgZnFe) and MgZnFe-Triazol LDH (MgZnFe-Tz) nanocomposite. Seventy-five wastewater samples were collected from a dairy farm sewage system. The sensitivity of isolated pathogens was tested against two commonly used disinfectants (Terminator and TH4) and was assessed against the three LDH samples at different concentrations. The overall prevalence of S. agalactiae, S. dysgalactiae and Staph. aureus was significantly at 80.0% (P-value = 0.008, X2 = 9.700). There was variable degree of resistance to the tested disinfectants, whereas the antimicrobial activity of CoZnFe LDH was increased significantly at a concentration of 0.005 mg/L followed by MgZnFe LDH while MgZnFe-Tz LDH showed minor antibacterial potency. It was concluded that CoZnFe LDH showed a better biocidal activity in killing the isolated resistant pathogens, making it a good choice tool in combating the zoonotic microbes in wastewater sources.


Materials and methods
Sample collection. A cross-sectional study was performed during the period from May to September 2021 in which seventy five wastewater samples were collected from a local dairy farm sewage system. Wastewater samples, which are alkaline in nature, were collected in sterile transparent glass containers, where the containers were sterilized in a hot air oven before collection. At the collection point, containers were rinsed several times with the water to be collected, filled, corked tightly and then labeled and sent to the laboratory of Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Beni Suef University in ice box and when required were stored transiently at 4 °C. All samples were examined immediatley after being recieved to avoid any possible physico-chemical changes in the wastewater samples 31 .
Isolation and identification of Staphylococcus and Streptococcus. Each wastewater sample was directly cultured on the surface of sodium azide crystal violet blood agar (Oxoid, CM0259) and Mannitol Salt agar (MSA) media (Oxoid, CM0085) for isolation of Staphylococcus and Streptococcus species, respectively. All plates incubated at 37˚C for 24-48 h. The colours and morphologies of the colonies were noted from the selective plates followed by the biochemical tests for identification of Staphylococcus and Streptococcus species 32 .
Molecular identification of the isolates. Firstly In-vitro evaluation of disinfectants and prepared nano-material efficacy against the isolated bacteria. The biocidal power of two commercially used disinfectants in water treatment were approved by the food industry 37 ; Terminator (glutaraldehyde(150 g/L) + quat. ammonium chloride(100 g/L)), TH4 (is a combination of 4 quaternary ammonium, glutaraldehyde and 2 terperne derivatives), both disinfectants were obtained from 6th October 3rd Industrial Area, Egypt. The disinfectants were tested using different concentrations of them against 60 strains of s. agalactia, s. dysgalactia and staph. aureus isolated from the wastewater samples using broth agar well-diffsuion method 38 .
Antibacterial activity assay. Antibacterial activity of all samples was assessed on all the identified bacterial isolates using agar well diffusion technique 39 . Inoculum containing 10 6 CFU/ml of each bacterial culture to be tested was spread on the surface of nutrient agar plates with a sterile swab moistened with the bacterial suspension. Subsequently, wells of 6 mm diameter were cut into the agar medium using sterile micropipette tips and filled with 40-50 μl of the tested materials and allowed to diffuse at room temperature for 2 h. The plates were then incubated in the upright position at 37° for 24 h. Wells containing the same volume of DMSO (10.0%), (Oxford -India) were used as received without any further purification. Sodium hydroxide (NaOH) was purchased from Egyptian Piochem for laboratory chemicals. The co-precipitation method was used to prepare ZnCoFe (LDH) following a procedure similar to our previous work 39 . The ratio of Zn nitrate to Cobalt nitrate to Ferric nitrate used was adjusted to be 1.5:1.5:1 by mole respectively. Briefly, metal nitrates were precipitated using a slow addition (0.1 mL/min) of NaOH solution (2 M) until the solution pH reached 9 to assure complete precipitation of the corresponding metal hydroxides. To follow, the precipitated hydroxides were left under continuous stirring overnight to age. The formed suspension was filtered and washed using distilled water and finally washed with ethanol.  .73 nm respectively. The decrease in the crystallite size after the addition of the triazol compound may be attributed to the capping of this compound to the layers of the MgZnFe LDH phase therefore preventing longrange order of such layers during the aging step of the precipitated LDH phase. FTIR spectra of all samples are shown in Fig. 2. As shown in Fig. 2a, the FTIR spectrum of the CoZnFe LDH sample shows a broad peak around 3400 cm −1 , which can be ascribed to the OH − stretching due to adsorbed water molecules 39 . Two sharp peaks at around 1510 and 1368 cm −1 can be attributed to the stretching mode of the nitrate ions 41 . The small peak at around 2900 cm −1 may originate from ethanol molecules used during the washing step 42 43,44 . These bonds originate from the di or tri metal center in the octahedron structures forming the LDH layers. The MgZnFe sample shows similar FTIR spectra to CoZnFe sample with one extra peak at 1630 cm −1 is can be attributed to the bending vibration of the interlayer H 2 O water molecules 45,46 . FTIR spectra of MgZnFe-Tz was similar to that of MgZnFe LDH with extra peak at 1230 cm −1 originating from the N-H bending of the amino group in the triazol (3-amino-1H-1,2,4-triazole) compound 47 .

Results and discussion
SEM images of all samples are shown in Fig. 3. CoZnFe LDH shows a typical layered structure as presented in Fig. 3a. Similarly MgZnFe LDH showed a similar morphology as shown in Fig. 3b. No change in morphology for the MgZnFe-Tz sample (Fig. 3c) was observed as compared to the MgZnFe LDH sample. Figure S1 shows TEM images for the prepared samples. These images show that the prepared LDH has a wide layer size distribution. This is attributed to the co-precipitation method that lacks control over the layer size while being a cost effective and simple preparation method suitable for practical applications.
Edx analysis of the samples is shown in Fig. 4. All Edx spectra did not show any foreign species, thereby reflecting the purity of the prepared samples. Sample CoZnFe LDH (Fig. 4a) showed peaks for Co, Zn and Fe. Similarly, MgZnFe LDH and MgZnFe-Tz (Fig. 4b,c respectively) showed peaks for Mg, Zn and Fe only. Figure 5 represents the nitrogen adsorption desorption isotherms of all samples. All isotherms can be classified as type IV isotherm with hysteresis loop of types H3 48 . Type IV isotherm originates from samples with mesoporous structures. Mesoporous materials are those with pore widths ranging between 2 and 50 nm 48 . Moreover, H3 hysteresis loops originate from non-rigid aggregates of plate-like particles 48  www.nature.com/scientificreports/ LDH samples. LDH samples has layered structure where the divalent and trivalent cations from alternative octahedrons where the oxygen atoms are bonded at the corners and the metal cation in the center. These octahedrons form the layered structure of the LDH samples leading to the H3 type loop in the nitrogen adsorption-desorption isotherm. Table 2 shows the BET surface area, pore volume and mean pore diameter of all samples. As shown MgZnFe LDH has almost 2.3 folds the area of CoZnFe LDH probably because of the larger crystallite size. After Tz addition, the surface area decreased by about 35% owing to the adsorption of Tz on the LDH surface.
Antimicrobial study. The presence of microbial pathogens in dairy farms' wastewater might cause the contamination of raw milk with these pathogens through wastewater one way or another, which might be of major public health significance therefore, it became a need to control these pathogens. Results in Table 3 the prevalence of some pathogens of zoonotic importance that might be found in wastewater such Staph. aureus S. agalactica and S. dysaglactica it showed high prevalence (80.0%) of collected samples were positive for microbial isolation. Staph. aureus was the dominant pathogen to be isolated (41.3%) followed by S. dysaglactica and S. agalactica (22.7 and 16.0%, respectively) this was statistically significant (P > 0.05). the isolated strains of Staph. aureus S. agalactica and S. dysaglactica were genetically assayed to detect 16S rRNA gene specific for S. agalactia and S. dysgalactia amplified 405 bp and 572 bp, respectively (Fig. 6 A), also 23S rRNA gene specific for Staph. aureus (B) amplified 1250 bp (Fig. 6B). Moges et al. 49 reported similar results to the findings in our study where he mentioned that (85.0%) of the collected wastewater samples were positive for one or two pathogens but recorded much lower isolation rate to S. aureus (8.2%) than our findings as well, Ben Said et al. 50 51 performed a study on fish cultured in lack ponds located near residential areas and agricultural waste disposal sites (water of low quality). The authors found the isolation of S. agalactica was (10.42%) which is nearly similar to our findings. In this study, the teamwork was concentrating on pathogens which are mainly related to dairy animals and industry, such S. dysaglactica, S. agalactica and Staph. aureus and we contribute the presence of these pathogens in the farm wastewater due to the measures being taken in the farm under the study like throwing contaminated milk and animal wastes in the farm sewer system.
Results illustrated in Table 4 showed that the sensitivity of the isolated strains of S. dysaglactica, S. agalactica and Staph. aureus was significantly high to the commonly used disinfectants in water treatment at different concentrations. Whereas there were variable degrees of resistance to the nanoparticles used, as it was cleared that CoZnFe LDH showed a significant efficacy in combating of S. dysaglactica, S. agalactica and Staph. aureus isolates followed by MgZnFe LDH and the lowest biocidal potency was showed by MgZnFe-Tz LDH. According to our findings, it was revealed that the elevated resistance rate to the used disinfectants was confirmed by determination of the QacED1 gene (Fig. 6C) responsible for the resistance to quaternary ammonia compound, the main constitute in the used disinfectants. www.nature.com/scientificreports/ Based on our findings, it was evident that LDH had a strong antibacterial effect on the screened microbes because bacteria were able to stick to and adsorb to its surface via electrostatic forces produced by the metallic ion exchanged LDH, leading to higher antibacterial efficacies 52,53 . Recent years have seen a lot of interest in LDH's antibacterial capabilities. LDH toxicity was evaluated by 54 against Streptococcus, with an EC50 of 10 mg/L following 72 h of LDH exposure. Additionally, the LDH concentration of 50 mg/L fully prevented the development of Streptococcus. Also, LDH exhibited a long-lasting antibacterial activity against both Gram-positive bacteria (Staph. epidermidis, S. pyogenes, and Staph aureus) as well as Gram-negative bacteria (Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Salmonella) as reported previously 26,55 .
It was obvious that all the examined microorganisms displayed an In-vitro considerable antibacterial activity against Zn-Fe LDH. This could be explained by the release of hydroxyl ions from the Zn-Fe LDH in a wastewater        www.nature.com/scientificreports/ setting. These hydroxyl ions are extremely reactive free radicals that have the potential to damage a variety of biomolecules, including the DNA and cytoplasmic membrane of bacteria as well as cause protein denaturation. These free radicals are believed to be released easier from layers of small layer sizes (Fig. 7a). These layers are believed to be easily dissolve compared to layers of large layer size. Additionally, the existence of various metal mixtures in LDH like Fe, Ni, Zn, Co are usefully reactive with proteins and preventing the controlled transfer through the plasma membrane by permeability affecting the transport system and lead to the death of bacteria 56,57 .
On the other hand, layer size can affect the attachment on the bacteria cell membrane. Zn-Fe LDH's have positive charge with zeta potential values up to 30-50 mV, improving its antibacterial action 58 . This may be explained by the abundant holes and high peptide poly-glycogen content of Gram-positive bacteria's cell wall, which made it easy for foreign molecules to enter the cell and facilitated the deadly effect-producing faster ion absorption 59 . It is believed that layers with larger sizes can contribute positively to the attachment mechanism compared to smaller sizes (Fig. 7b). To sum up, the prepared samples have a wide layer size distribution. Small layers easily dissolve and produce free radicals and metal ions. On the other hand, large layers easily attach on cell membrane causing membrane disruption and deterioration. Both size contribute to the observable antibacterial effect of the prepared LDH samples.

Conclusion
Since there is a global trend toward using treated wastewater and due to the scarcity of water resources all over the word, therefore it became a need to search for alternatives to traditionally used materials that are used in the treatment of different contaminants in wastewater such bacteria, heavy metals and antibiotics. Three Zn-Fe based LDH samples were successfully prepared and characterized using XRD, FTIR, SEM, BET and Edx. The prepared samples were tested for their antibacterial properties against pathogens in real dairy wastewater samples. From the current study, it was concluded that S. agalactiae, S. dysgalactiae and Staph. aureus prevailed in dairy farm wastewater sources. The lack of proper sanitation for such wastewater effluents will result in the in contamination of dairy products. The use untreated wastewater can result in the infection of dairy animals and/ or their products through the capacity of the pathogens that could gain access to human food chain through the consumption of contaminated products. Due to the acquired resistance of most pathogens to commonly used disinfectants. Therefore, it is important to regularly clean and remove organic matter from entering the animal's water sources and passing through the entire system of dairy production. The better efficacy of CoZnFe LDH has been demonstrated in this study, making them a promising material in controlling the zoonotic pathogens in water sources to improve the dairy industry.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.